HCV, the primary etiological agent of chronic non-A, non-B hepatitis is a genetically heterogeneous group of viruses classified into multiple distinct genotypes and subgenotypes. HCV infection is characterized by persistent viremia and often progressive liver disease. Chronic hepatitis C is the most common indication for liver transplantation in adults, and at the University of Washington Medical Center, more than one third of transplant candidates have evidence of active HCV infection. Despite its widespread occurrence and clinical importance, the pathogenesis of chronic hepatitis C is not well understood. A great impediment to the understanding of the relationship between HCV infection and disease development has been the lack of a appropriate tissue culture system as well as mouse or other small animal models which are permissive to viral infection. In addition, it has been difficult to establish correlations between the extreme mutational variability of the virus and clinicopathologic outcomes of chronic hepatitis C mostly because of the long natural history of the disease and the paucity of detailed longitudinal studies. This application represents the combined efforts of investigators with expertise in clinical virology (HCV quantitation and subspecies characterization), the development of differentiated hepatocyte cell lines and liver growth regulation, the establishment of transgenic mouse models for hepatocyte transplantation and gene transfer techniques. The general objectives of the project are to develop tissue culture and animal model systems to study major questions of hepatitis C pathogenesis and therapeutics and to use in these studies virus isolates which have been well characterized as to genotype, mutational variability and relationship to disease progression obtained from population cohorts enrolled in longitudinal studies. The proposal is constituted by four sections and one core facility organized as follows: 1) CLINICAL VIROLOGY, to determine whether HCV replication correlates with disease outcome and to test the hypothesis that HCV genetic determinants may influence the clinicopathologic outcome of chronic hepatitis C in humans; 2) TISSUE CULTURE MODELS, to establish differentiated, replicative human hepatocyte cell lines to study direct HCV effects on hepatocyte survival, growth and transformation as well as to analyze HCV mutability in the absence of immunologic selective pressure; 3) ANIMAL MODELS, to develop transgenic mice with mouse/human hepatocyte hybrid livers which should be permissive to HCV infection and may serve as a suitable model to study HCV infection and disease; 4) GENE THERAPY, to develop new strategies against HCV using ribozyme gene transfer; 5) MOLECULAR VIROLOGY CORE, to provide well characterized HCV isolates and quasispecies from human samples for studies in tissue culture and animal models, and to assess viral persistence and infectivity in cultured hepatocytes and transgenic hybrid mice. These studies are expected to contribute in fundamental ways to the understanding of the pathogenesis of hepatitis C and to devising novel approaches for treatment.